Antibodies For discrimination of prions

ABSTRACT

In the present invention, we described the use of anti-DNA antibody for the detection of prions and diagnosis of Transmissible Spongiform Encephalopathies (TSE) diseases in animals and humans.

FIELD OF THE INVENTION

[0001] In the present invention, we described the use of anti-DNAantibody (also referred to hereinafter as “an anti-nucleic acidantibody”) for the detection of prions and diagnosis of TransmissibleSpongiform Encephalopathies (TSE) diseases in animals and humans.

BACKGROUND OF THE INVENTION

[0002] Transmissible spongiform encephalopathies (TSEs) comprise a groupof rapidly progressing, neurodegenerative fatal diseases that affectboth humans and animals. TSEs have clinical and neuropathologicalcharacteristics which include devastating dementia, pyramidal andextrapyramidal signs with myoclonus, multifocal spongiform changes,astrogliosis, amyloid plaques, neuronal loss, absence of inflammatoryreaction and are usually characterized by a long incubation period.

[0003] In animals, a commonly known example of TSE disease recognizedfor over 200 years, is scrapie, which is found in sheep and goats(McGowan 1922). Other animal TSE diseases have also been described, suchas transmissible mink encephalopathy (TME, Marsh 1976), chronic wastingdisease of mule deer and elk (C W D, Williams 1980), bovine spongiformencephalopathy (BSE, commonly known as “mad-cow” disease (Wells 1987),and the more recently described feline spongiform encephalopathy ofdomestic cats, pumas, and cheetahs (Wyatt 1991).

[0004] In humans, TSEs have been traditionally classified intoCreutzfeldt-Jakob disease (CJD), kuru, Gerstmann-Straüssler-Scheinkersyndrome (GSS) and fatal familial insomnia (FFI). Among them, Kuru hasbeen described only in the Fore linguistic group of New Guinea. For manyyears after its first recognition in 1957, kuru was the most commoncause of death among women in the affected population, but itsoccurrence has declined because of the cessation of cannibalism that hadfacilitated disease transmission. As of today, only a few cases stilloccur due to the long incubation periods typical of this condition.

[0005] Although these rare neurodegenerative disorders occur in about0.5 to one person per million worldwide (Brown 1987), TSEs attractedconsiderable public attention because of the unique biology and concernsabout a onset of the epidemic of a newly recognized bovine spongiformencephalopathy (BSE) and its potential effects on human. There ismounting evidence that through dietary exposure to BSE infected tissues,it has poses a serious threat to public health and has resulted in anincreased number of incidents of a newly recognized variant form of CJD(vCJD). Until now, there have been more than 100 cases of vCJD reported,a majority which are located in UK.

[0006] It is believed that prions are the pathogenic agent causing TSE.Many efforts have been directed towards identifying the etiologicalagent that causes TSEs. Early on, the transmissibility of TSE diseasehad been experimentally demonstrated in many cases, kuru and CJD fromhumans to chimpanzees (Gajdusek 1966, Gibbs 1968), transmissible scrapiefrom sheep to sheep (Cuillé 1936) and across species to goat (Pattison1957). The most significant breakthrough was the successful transmissionof scrapie to mice, by Richard Chandler in 1961 (Chandler 1961).Chandler's discovery greatly facilitated TSE research by providing anexperimental model that was cheaper and easier to manipulate. Althoughall of the above modes of transmission were demonstrated experimentally,the cause of recent BSE in cattle and new variant CJD in human (vCJD)was considered a consequence of dietary exposure to the mix of scrapiesheep carcasses rendered for animal feed in the case of BSE (Brown1997), and to beef from cattle affected with BSE in the case of vCJD(Bruce 1997).

[0007] It was suggested that TSE diseases might be caused by “slowviruses” or viroids (Gajdusek 1977). However, the extreme resistance ofscrapie infectivity to radiation, nucleases, and other reagents damagingto genetic materials are inconsistent with the “virus” theory. Moreover,the infectious TSE agent could tolerate very high levels of heat andhigh concentrations of formaldehyde (Pattison 1965) while still able toreplicate with the ‘incubation period’0 varying from a few months toover a year (Alper 1966).

[0008] All these “unusual” characteristics of the TSE infectious agentled Dr. Stanley Prusiner to propose the concept of “prions” in 1982(Prusiner 1982). Prion (PrP), which stands for nucleic acid-freeproteinaceous infectious particle, is a glycoprotein present in humansand animals. In humans, it is encoded by PRNP on chromosome 20 (Robakis1986). The cellular form of this protein (PrP^(C)) has two N-linkglycosylation sites and a GPI anchor at the C-terminus. It has been mostcommonly found in neurons, and, to a much lower extent, it has also beenfound in other cells such as leucocytes, monocytes and platelets (Holada2000). Furthermore, a soluble form of PrP that lacks the glycolipidanchor was detected in murine and human serum. The transmissible scrapiedisease form of the prion protein (PrP^(Sc)) is a protease resistantisoform of its cellular precursor and is predominantly found in brain.At much lower level, it has also been found in tonsil, spleen, and lymphnodes in vCJD patients (Parizek 2001). The conversion from PrP^(C) toPrP^(Sc) is believed to be accomplished through a conformational changewithin the protein. Although there is still ambiguity concerning themechanism of the conversion, much experimental evidence indicates thatin the presence of PrP^(Sc), normal PrP^(C), acting as a substrate,undergoes a conformational structure change, and becomes PrP^(Sc). Thisprocess of propagation involves replicating the conformation of PrP^(Sc)in PrP^(C) and results in PrP^(Sc) aggregation and amyloid rodformation, hence causing cell death (Hope 1986, Horwich 1997). As aresult of Prusiner's-concept of the “prion” as an infectious agentresponsible for scrapie disease, and by extension, that of all TSEdiseases gave rise to the notion of what are commonly referred to asPrion diseases to describe a class of pathologies believed to be linkedto this protein.

[0009] Characteristics of PrP^(C) and PrP^(Sc)

[0010] The major property that differentiates PrP^(C) and PrP^(Sc) istheir distinct conformation. The structural change from PrP^(C) toPrP^(Sc) is most supported by a crucial conformational change, involvinga substantial increase in the amount of beta-sheet structure of theprotein, with possibly a small decrease in the amount of alpha-helix,indicated by circular dichroism and infrared spectroscopy (Pan 1993,Caughey 1991). The solution structure of a fragment of the mouse PrP^(C)has allowed a direct determination of secondary structure content of aportion of PrP^(C) (121-231) by NMR (Riek 1996).

[0011] Protease resistance is another characteristic that distinguishesPrP^(Sc) from PrP^(C). In cultured cells and brain or in samples frommany patients with GSS, PrP^(Sc) is smaller than its cellular precursorPrP^(C). Even though cellular prion and scrapie prion are two isoform ofsame PRNP genomic product, PrP^(C) is completely degraded by ProteinaseK treatment while PrP^(Sc) undergoes only limited digestion. Thedigestion yields a form of protein referred to as PrP 27-30 in which theN-terminus has been removed. PrP 27-30 has been postulated to be thePrP^(Sc) core required for PrP^(C) hosted PrP^(Sc) replication. Theprotease treated prion molecule, PrP 27-30 or PrP^(res), is tightlylinked to scrapie infectivity (Gabizon 1988), and provides additionalevidence that PrP^(Sc) is an infectious protein.

[0012] An additional attribute, perhaps linked to the significantincrease in β-sheet structure and concomitant protease-resistance, isthe observed difference in solubility between PrP^(Sc) and PrP^(C).While PrP^(C) is a soluble protein, the PrP^(Sc) isoform is highlyinsoluble. Furthermore, PrP^(C) is found attached to the surface ofneurons through a GPI tail anchored into membrane (Shyng 1994) whilePrP^(res) is found in the cytoplasm of affected cells (Taraboulos 1990),most likely associated with late endosomal and lysosomal compartments(Arnold 1995), and PrP^(Sc) is also localized in amorphous aggregates inenriched fractions from infected brain (Meyer 1986). Interestingly, adisease-associated mutant PrP, the PrP^(159stop) mutant was foundexclusively in nucleus (Lorenz 2002).

[0013] There is mounting evidence indicating a tight linkage betweenscrapie infectivity and PrP 27-30. Even in the purest samples, theestimated ratio of PrP molecules to infectious units is ˜10⁴ to 10⁵(Horwich 1997, Bolton 2001). At such low levels of infectivity, it ispossible that other components, co-factors, or covalent modifications,are required for infectivity. The transgenic studies on thesusceptibility of mice expressing chimeric human-mouse PrP^(C) suggestthe presence of at least one host factor other than PrP^(C), tentativelytermed factor X, which might function as a molecular chaperone in theformation of PrP^(Sc) (Telling 1995).

[0014] Other Molecules Associated with Prion Pathogen

[0015] About 15 to 20 strains of scrapie have been identified based ontheir incubation period and lesion patterns in the inbred mice. After aserially inoculation passage in inbred mice homozygous for a single PRNPgenotype, all the scrapie strains retained their original diseaseprofile. These observations led investigators to question whether variedphenotypic strains were dominated by different conformation isoforms ofsame cellular prion precursor, a possibility suggested byconformation-dependent immunoassay (Safar 1998), or whether thesestrains were a result of various PrP^(Sc) associated molecules.

[0016] Many researchers have identified various nonprotein moleculesthat are bound to prion proteins. The precise biological andphysiological roles remain the topic of further investigation. Copperand zinc have been demonstrated to bind to PrP^(C). In vitro, thesedivalent metals may contribute to prion superoxide dismutase (SOD)-likeactivity. Such SOD-like activity and copper content are dramaticallyreduced in scrapie-infected brain (Wong 2001).

[0017] In addition, prion rods, composed mainly of insoluble aggregatesof the N-terminally truncated prion protein (PrP 27-30) are found to beassociated with 1,4-linked glucose units. Sphingolipids, polysaccharideand other membrane components were also found in prion aggregates (Appel1999, Klein 1998). The interaction between prion protein and lipidmembranes could play a role in PrP conversion. For example, thenegatively charged lipid membrane-inserted conformation of PrP is richerin β-sheet structure while the binding of PrP to raft-like membranesinduces the formation of α-helical structure (Sanghera 2002).

[0018] In early 1990's, Snow et al, studyingGerstmann-Sträussler-Scheinker syndrome, Creutzfeldt-Jakob disease andscrapie, have documented the association of sulfated proteoglygan to theprion protein amyloid plaques (Snow 1990). In an immunohistochemistrystudy using heparan sulfate antibodies (anti-HS) and heparan sulfateproteoglycan antibodies (anti-HSPG), McBride has demonstrated thecorrelation and association between HSPG and abnormal PrP inscrapie-infected mice brain. This correlation and association wasobserved as early as 70 days post-infection and throughout the course ofthe disease (McBride 1998). In in vitro conversion from PrP^(C) toPrP^(Sc) and in prion infectivity reconstitution experiments, sulfateglycans have been shown either to facilitate the conversion or toescalate infectivity (Wong 2001, Shaked 2001a). With recombinantGST::full-length prion and GST::prion fragment, Warner recentlydemonstrated direct binding of recombinant prion to heparin and heparansulfate (Warner 2002). The peptide region 23-52 in prion sequence waspositive in all HS and HSPG binding tests. Since the peptide failed tocompete with full-length prion for binding to heparin, the authorsuggested that there might be another major GAG-binding site in intactPrP^(C). Another noteworthy observation is that GAGs from differentspecies (bovine and porcine) or from different organs (lung, kidney andintestine) have shown different affinities for prion binding. Thedifference in affinity may be due to prion sequence itself, or maydepend on the presence of particular sugar unit in the tested GAGs.Through a mechanism that is perhaps different from that by which glycansparticipate in the conversion of PrP^(C) to PrP^(Sc), DNA could alsoconvert cellular prion protein into β-sheet conformation (Cordeiro2001). Nandi demonstrated that prion peptide 106-126 is the region thatparticipated in the nucleic acid-prion complex association (Nandi 1998).Interestingly, not only was PK resistant amyloid aggregate obtained fromthe interaction between prion protein and nucleic acids, the nucleicacid morphology also changed to condensed globular structures, similarto nucleic acid structures induced by the HIV-1 NCp7 protein, but not tothe structure induced by histones (Nandi 2001). Based on those in vitroconformation and conversion studies, it was hypothesized that DNA wouldact as a guardian of the PrP^(Sc) conformation as well as a catalyst tofacilitate PrP^(Sc) conversion and aggregation (Cordeiro 2001).

[0019] Whether one accepts or rejects the “protein only” or “prion only”hypothesis, the effort to link inherited information to TSE disease orthe search for genetic make up related to TSE disease has never stopped.The presence of a tightly bound RNA or DNA molecule in the prionparticle was proposed to explain propagation of different strains ofscrapie agent with distinct phenotypes in animals homozygous for thePRNP gene (Weissmann 1991). Analysis of highly purified scrapie prionsby return refocusing gel electrophoresis revealed the small size ofremaining nucleic acids, although the size of extracted nucleotides wastoo small to encode any meaningful protein (Kellings 1992). In a recentreport, however, Narang indicated that animals inoculated with ssDNApurified from scrapie-hamster brains mixed with non-pathogenic priondeveloped clinical disease (Narang 2002). Based on his findings, hepostulated that the “accessory protein” coded by the ssDNA may beinvolved in PrP^(C) to PrP^(Sc) conversion. Although the role of nucleicacids in prion-associated disease is controversial, it is clear thatPrP^(Sc) aggregates are tightly associated with these small molecules.

[0020] Infectivity and Transmissibility of Prion Diseases

[0021] Classic CJD in human has been grouped into three etiologicaltypes: sporadic (CJD), inherited (GSS or FFI), and acquired, which isvery rare and includes diseases such as kuru and iatrogenic CJD. Thereis no hard evidence indicating any of CJD diseases is related to animalTSEs that may have crossed species barriers. The epidemic of kuru hasprovided the largest body of evidence of acquired human prion disease.Searching for risk factors and possible sources of infection in sporadicCJD patients revealed no significant correlation of disease to diet,blood transfusion or receiving other blood product. However, afterintracerebral inoculation to mice, the infectivity in blood obtainedfrom CJD patients indicated the possible presence of the CJD agent(Manuelidis 1985, Tateishi 1985).

[0022] BSE appears to have originated from dietary exposure. Nutritionalsupplements of processed meat and bone meal derived from scrapie diseaseinfected carcasses were used to feed cattle livestock and other captiveanimals. In spite of BSE originating from scrapie, no case of de novoinfection or cow-to-cow transmission has been reported.

[0023] There is mounting evidence, however, that links vCJD to BSE, Thegrowing epidemiological data locates the majority of vCJD cases in UKwhere the overwhelming majority of BSE cases have also been reported.The link between vCJD and BSE is further supported by theneuropathologic evidence obtained from BSE-adapted macaques, the nearestmodel to humans (Bruce 1997), and from the study on inbred miceinoculated with the agent causing BSE and vCJD (Lasmézas 1996).

[0024] Although no vCJD patient has been documented as a victim ofhuman-to-human transmission, the close link between BSE and vCJDattracted considerable attention. Concerns about human infection havebeen based on the observation that PrP^(Sc) is readily detectable in BSEand vCJD lymphoreticular tissues but not in classic CJD (Hill 1997),followed by the presumption that scrapie pathogen from sheep passage tocattle may have altered host range and become more adaptable to human.Experimental precedents for such behavior are well known: passage ofmouse-adapted strains of scrapie through hamsters altered theirtransmissibility on back passage to mice (Kimberlin 1987, Kimberlin1989); human strains of kuru or CJD did not transmit to ferrets or goatsuntil passaged through primates or cats (Gibbs 1979); and a bovinestrain of BSE did not transmit to hamsters until passaged through mice(Foster 1994). Alternatively, if BSE originated from a spontaneousmutation in cattle, experimental studies of species susceptibility tothis new strain of transmissible spongiform encephalopathy (TSE) had notsufficiently advanced to predict that humans would not be susceptible.

[0025] In addition to CJD infectivity in blood described above, otherTSE infectivity in blood has also been demonstrated in variousexperimental animals. Most blood for infectivity studies was obtainedfrom TSE -adapted rodents such as mice and hamsters. The only exceptionwas a study conducted in the sheep model. In this experiment, a sheeptransfused with whole blood, taken from another sheep inoculated withBSE brain lysate, developed symptoms of BSE (Houston 2000, Hunter 2002).However, these experimental results yet need to be fully evaluated. Theinfectivity in blood has been established in rodent animals throughintracerebral and intravenous transmission with mice-adapted BSE,mice-adapted vCJD and other rodent animal adapted TSE strains. Althoughthe infectivity in lymphocyte-rich buffy-coat is greater than in plasma,it only accounts for relatively a small portion when compared to wholeblood inoculums. The molecular definition of this infectious agentpresent in the blood is still under investigation. It is anticipatedthat finding of such infectious agent in blood would help us to betterunderstand the relationship between PrP^(Sc) and TSE disease.

[0026] Study on human CJD and vCJD disease indicated that genomicsusceptibility may yet be another factor that may influence the spreadof TSE in humans. The majority of sporadic CJD patients were found to behomozygous for Met/Met or for Val/Val at codon 129 (Belay 1999).Nevertheless, all reported vCJD cases have been found to be homozygousfor Met/Met.

[0027] The size and duration of vCJD epidemic still remains uncertain.Depending on the assumptions made and the modeling calculationsemployed, different predictions were proposed. One estimation of totalvJCD predicts as few as 205 cases (Valleron 2001). On the other hand,another prediction for vCJD mortality for the next 80 years ranges from50 to 50,000 if infection comes only from BSE. It could reach up to150,000 if BSE is proven to infect sheep and if subsequently it isallowed to enter human food chain (Ferguson 2002). Although it isimpossible to make accurate predictions if the necessary parameters areeither mistaken or not available, one thing is certain that if vCJDinfectivity is present in blood, any prediction will be anunderestimate. In addition, vCJD has been proven to be a new diseaseentity and not simply the result of increased surveillance of CJD inhumans (Hillier 2002).

[0028] Countermeasures have been taken by government to eliminate thespread of BSE incidence. Ruminant protein feed was banned in US and UK(1988). A series of measures have also been taken to prevent potentiallyinfected meat from entering human food chain. To further reduce thehuman risk, FDA and CBER has issued a new policy in Aug. 2001, whichindefinitely defers any human blood donor who stayed cumulative ≧6 monthduring 1980-1996 in the United Kingdom (FDA 2001).

[0029] Diagnostic Assay for Prion Disease

[0030] Clinical symptoms of prion disease often overlap with those ofother neuronal degenerative diseases that make diagnosis difficult. Sofar, PK resistant PrP 27-30 is the only protein marker linked to TSEdisease. Therefore, the detection of this agent has become the focus ofassay development. However the development of monoclonal antibodyspecific for PrP^(Sc) was extremely difficult, not only becausepathogenic PrP^(Sc) isoform and normal cellular PrP^(C) are twoconformers of the same protein with an identical primary sequence, butalso because the prion appears to be a weak immunogen. The only antibodyreported to be able to recognize PrP^(Sc) specifically is notpractically useful (Korth 1997). Other prion sequence-specificmonoclonal and polyclonal antibodies are unable to distinguish PrP^(Sc)from PrP^(C). Nevertheless, these antibodies (such as 3F4, 6H4 describedin US4806627 and EP0861900.) are still commonly in use for capture orfor detection of prion protein in combination with sample treatment andseparation techniques to isolate PrP^(Sc) from PrP^(C) (Korth 1997,Kascsak 1987).

[0031] Since the outbreak of BSE in 1986, all commercially availabletests for prion disease use, as their sample source, tissues taken frompostmortem animals and humans. Among those, a tissue homogenate-basedPrP^(Sc) assay, referred to as DELFIA (dissociation-enhanced lanthanidefluoroimmunoassay), was developed for the detection of scrapie prion(Barnard 2000 and a method described in US20020137114A1). It requires aprotein denaturation step using GdnHCI, in combination with optionalsample PK treatment and PrP^(Sc) enrichment by sodium phosphotungsticacid (NaPTA) precipitation. Since the transformation of PrP^(C) toPrP^(Sc) is accompanied by the burial of epitopes near the N terminus ofPrP, in DELFIA, monoclonal antibodies directed against the N-terminus ofPrP are used to measure the difference of mAb binding affinity to theα-helical and β-sheet conformations before and after PrP denaturation(Peretz 1997). Another conformational-dependent immunoassay (CDI)combined with ELISA and fluorescence detection (Safar 1998, US20010001061A1, US20020001817A1) was described in conformation studies inPrP^(Sc) strains.

[0032] In a tissue distribution study of PrP^(Sc) in vCJD patients, animproved NaPTA precipitation was described to enrich PrP^(Sc) from brainand from other peripheral tissue homogenates (Wadsworth 2001

[0033] ). The modification employed endonuclease treatment to reducesample viscosity prior to NaPTA precipitation. The recovery of PrP^(Sc)in the precipitated pellet was reported to be consistently greater than90% while recovery of PrP^(C) was about 5%. After PK digestion, thepresence of PK resistant prion was verified in Western blot using 3F4monoclonal antibody.

[0034] In another similar immunoblot assay, P K digestion was also usedto eliminate PrP^(C). 6H4 was then used to determine the presence ofPrP^(Sc) (Schaller 1999). Based on this first generation assay, asecond-generation luminescence immunoassay was developed in which 6H4was coated on plates as a capture antibody. The horseradish peroxidase(POD)-conjugated detection antibody used was a mouse monoclonal anti-PrPantibody, able to form a complex with PrP27-30 bound to 6H4 (Biffiger2002).

[0035] The European Commission in 1999 evaluated 4 BSE test kits fromdifferent manufacturers (Moynagh 1999). They all used bovine braintissue as a sample source, and all required a separate samplepreparation procedure. Depending on the kit instructions, the braintissue homogenate needed to be processed, including denaturation, PKdigestion or PrP^(Sc) enrichment. The assay detection systems employedin DELFA, immunoblot, or in plate ELISA formats used eitherchemiluminescent or a colorimetric substrate.

[0036] In order to control the spread of the disease in the absence of alive-animal screening test, an extensive slaughter of cattle was carriedout once an affected animal was identified within a herd. The urgencyfor a live animal diagnosis assay was reinforced when the first cases ofvariant Creutzfeldt-Jakob disease was reported in 1996.

[0037] Antemortem TSE diagnosis development presents three majordifficulties: (1) insufficient sensitivity—Except in brain tissue,PrP^(Sc) concentrations in other tissues or fluids is considered to bevery low. Therefore, a highly sensitive technique is required fordetection. (2) Appropriate sample treatment—Any protein denaturation orPK digestion process may have a potential impact on pathogenic PrP^(sc)structure, with the possibility of causing a false negative result. Forexample, it has been suggested that an intermediate form of PrP^(Sc) maynot be PK resistant (Horiuchi 1999, Jackson1999, Swietnicki 2000). And(3), the lack of PrP^(sc)-specific antibodies and the incompletelycharacterized molecular relationship between the pathogenic agent andPrP^(Sc) in blood make it difficult to design an assay format forantemortem diagnosis.

[0038] A possible approach to boost the sensitivity is in-vitroamplification of PrP^(Sc). It has been reported that when PrP^(Sc) waspresent, repetitive cycles of sonication could induce protease-sensitivecellular PrP to form protease resistant aggregates. The authorsexplained that in this “protein-misfolding cyclic amplification” (PMCA)process, sonication could disrupt newly formed aggregates and generatemultiple smaller units for the continued formation of new PrP^(Sc)(Saborio 2001, WO0204954). At the end of 40 PMCA cycles, the sample wassubjected to PK digestion and detected by immunoblot. It claimed thatthe amplification generated more than 30-fold protease resistant PrP.Since proteinase resistant PrP were generated at the expense of thenormal prion protein as substrate through amplification cycles, a largequantity of same-species normal prion was required. It has not beendemonstrated whether normal prion from another species could also workas substrate, or prion protein from a recombinant source or from sourcesother than brain tissue could be used. Such evidence would be usefulwhen detection of vCJD is desired.

[0039] Immunohistochemistry of third eyelid lymphoid tissue has beendescribed for preclinical diagnosis of ovine scrapie (O'Rourke 2000,US6165784, US6261790). Relying on a small surgical procedure, the assaymakes use of sheep peripheral tissue, the third eyelid lymphoid forscrapie detection. The immunohistochemistry used a cocktail ofpan-specific monoclonal antibodies to differentiate one isoform from theother. Following formalin fixation to reduce PrP^(C) reactivity, thesample is subjected to formic acid and heat pretreatments which enhancethe PrP^(Sc) reactivity. In spite of the fact that the assay is stilltissue based and the observation that PrP^(Sc) displayed poorimmunoreactivity in immunohistochemistry staining unless treated withdenaturing agents, this antemortem preclinical diagnosis has made a steptowards live-animal test as well as provided a way of identification ofscrapie-affected sheep during the early, preclinical stage of scrapie.

[0040] In addition to the traditional identification of pathogenic prionby eliminating cellular prion followed by non-discriminatory anti-prionantibody recognition, other reagents were found to be able todifferentiate PrP^(Sc) from PrP^(C), such as plasminogen and fibrinogen.The mechanism of interaction between these human blood componentproteins and PrP^(Sc) is not clear. However, when immobilized onmagnetic beads, plasminogen selectively precipitated PrP^(Sc) from brainhomogenates of mouse, human, cattle and sheep. The evidence providedsuggested that a property common to PrP^(Sc) of various species, ratherthan prion primary sequence or the specific tertiary structure ofindividual PrP^(Sc) molecules, could be responsible for binding toplasminogen (Fischer 2000, Maissen 2001). The application for the use ofplasminogen and other serum/plasma proteins for the capture anddetection of pathogenic prion protein has been described in WO0200713and in US20010053533A1 (Aguzzi 2001).

[0041] Recent investigations have identified a new isoform of the prionprotein in the urine of animals and humans with prion disease (Shaked2001b, WO0233420A2). This isoform, referred to as UPrP^(Sc) by theinvestigators, was precipitable, PK resistant, and detectable only ininfected individuals but not in normal controls. Most importantly, asindicated in their publication, UPrP^(Sc) appeared long before theclinical signs developed in inoculated hamsters. However, when UPrP^(Sc)isolated from scrapie hamster urine was inoculated back in normalhamster intracerebrally, it did not cause disease even after 270 days,well beyond the incubation period in which animal would develop clinicalsigns if comparable amount of brain derived PrP^(Sc) had beeninoculated. It is not impossible that those hamsters, inoculatedintracerebrally with UPrP^(Sc), were still in a subclinical or carrierstate. Moreover, PK-resistant PrP was not found in the kidneys, whichimplies that this UPrP^(Sc) could have originated from other organs andbeen transported to the urine via the blood. This important observationwill undoubtedly lead to a better understanding of PrP metabolism.

[0042] Therefore there remains an unmet need for a better way to detectPrP^(Sc) and diagnose TSE in humans and animals. The aim of the presentinvention is to provide a non-intrusive way to isolate, concentrate andmonitor the TSE disease-related pathogenic prion protein. The invention,including the use of selective anti-DNA antibody to bind the PrP^(Sc)through recognition of an associated binding partner, involves thediscriminatory capture of PrP^(Sc) but not cellular prion protein. Weprovide evidence of a high affinity association of nucleic acid toPrP^(Sc), and we demonstrate that such nucleic acids::PrP^(Sc) complexsurvived even after PK digestion and nuclease treatment.

SUMMARY OF THE INVENTION

[0043] The evidence provided in support of this invention demonstratedthat PrP^(Sc) is associated with high affinity to nucleic acid, mainlyDNA as investigated. A similar association with nucleic acid was notobserved with normal cellular PrP^(C). The evidence also demonstratedthat the association was strong, resistant to PK digestion and nucleasetreatment, and that PrP^(Sc) could be readily isolated by selectiveanti-DNA antibodies.

[0044] This invention relates to the use of anti-DNA antibodies tocapture PrP^(Sc) through nucleic acids associated with high affinity toPrP^(Sc), in combination with any prion sequence-specific antibody forthe detection of PrP^(Sc).

[0045] In another aspect, this invention relates to the selectiveanti-DNA antibody, as described above, that preferably binds topathogenic prion protein but not to the normal cellular form of prionprotein.

[0046] In another aspect, this invention relates to the selectiveanti-DNA antibody, as described above, for the detection of PrP^(Sc)through high affinity recognition of associated nucleic acids incombination of prion sequence specific antibodies.

[0047] In another aspect, this invention relates to the selectiveanti-DNA antibody, as described above, for the isolation, purification,capture, elimination and monitoring PrP^(Sc) in biological reagentproduction.

[0048] In another aspect, this invention relates to compositions andkits for determining the presence of PrP^(Sc), comprising anti-DNAantibody, as described above, for either capture or for detection stepin the assay procedure.

[0049] In another aspect, this invention relates to compositions andkits for determining the presence of PrP^(Sc) antibody produced inresponse to high affinity associated DNA as a binding partner topathogenic prion protein.

[0050] In yet another aspect, this invention relates to anti-PrP^(Sc)antibodies and their production using the said nucleic acids that caninteract with and/or associate to PrP^(Sc), and their use in detectingnucleic acid::PrP^(Sc) complex and prion disease infection.

[0051] In another aspect, this invention relates to a non-harsh sampletreatment procedure involving nuclease digestion for the benefit of theuse of selective anti-DNA antibody as described above.

[0052] Some examples of specific embodiments of the invention are asfollows:

[0053] A method for discriminating between infectious and noninfectiousprions comprising:

[0054] first contacting a sample with an anti-nucleic acid antibody,

[0055] then adding a prion specific antibody to form a complex betweenthe anti-nucleic acid antibody, prion and prion specific antibody, and

[0056] detecting the complexes.

[0057] A method for diagnosing transmissible spongiform encephalopathiesin a patient comprising:

[0058] drawing a sample from a patient,

[0059] contacting a sample with an anti-nucleic acid antibody,

[0060] then adding a prion specific antibody to form a complex betweenthe anti-nucleic acid antibody, prion and prion specific antibody, and

[0061] detecting the complexes, whereby detecting the complexes providesand indication of transmissible spongiform encephalopathies in apatient.

[0062] An immunoassay for detecting infectious prions comprising:

[0063] providing a solid support having bound thereto an anti-nucleicacid antibody,

[0064] contacting the solid support with a sample,

[0065] washing the support to remove any unbound sample,

[0066] contacting the solid support with a prion specific antibody, andcarrying out a detection step to determine if prions are bound to thesolid support.

[0067] A kit for the detection of infectious prions comprising

[0068] a solid support having bound thereto an anti-nucleic acidantibody, and

[0069] a labeled prion specific antibody.

[0070] An immunoassay for detecting infectious prions comprising:

[0071] providing a solid support coated with an agent to bind ananti-nucleic acid antibody contacting the solid support with a sample,

[0072] washing the support to remove any unbound sample,

[0073] contacting the solid support with a prion specific antibody, and

[0074] carrying out a detection step to determine if prions are bound tothe solid support.

[0075] Another embodiment of the immunoassay described above provides asolid support coated or carrying an agent that is capable of binding theanti-nucleic acid antibody. For example, using avidin or streptavidin onthe solid support and biotinylating the anti-nucleic acid antibody sothat it binds to the solid support via the avidin or streptavidin.

[0076] A further embodiment of the invention is directed to a vaccinecomposition comprising anti-nucleic acid antibodies and apharmaceutically acceptable carrier.

[0077] A method of treating a prion disease in a patient comprisingadministering a therapeutically effective amount of a vaccinecomposition comprising anti-nucleic acid antibodies and apharmaceutically acceptable carrier.

[0078] A method of inducing neutralized infectious prions in a patientsusceptible to or suffering from a prion disease comprisingadministering a therapeutically effective amount of a vaccinecomposition comprising anti-nucleic acid antibodies and apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079]FIG. 1: anti-DNA IP capture and immunoblot of scrapie hamsterPrP^(Sc). Hamster scrapie PrP^(Sc) was immunocaptured by anti-DNAantibodies followed by SDS-PAGE and 3F4 immunoblot. Results wereobtained from three separate experiments (Lane# 1-4, 5-8 and 9-10).

[0080]FIG. 2: IP capture and immunoblot of BSE PrPSc. BSE PrP^(Sc) wasdetected by anti-DNA antibodies and DNA binding protein throughimmunoprecipitation (IP) followed by 6H4 immunoblot. Results wereobtained from two separate experiments (Lane# 1-11, and 12-14).

[0081]FIG. 3: IP and immunoblot of hamster prion with various treatment.IP and immunoblot were performed under various conditions for eachanti-DNA antibody. (1) Standard: IP performed in IP buffer with scrapieor normal hamster brain homogenate (lane 1, 5, 9,13,17-20). (2) DNAinhibition: 5 ug/mL phenol-chloroform extracted, ethanol precipitated,and sonicated Salmon DNA (Sigma, Mo., USA, Cat.# D7656) was added in theIP buffer as inhibitor (lane 2, 6,10,14). (3) Proteinase K digestion:scrapie hamster brain homogenate was treated with Proteinase K at 50ug/mL at 37 C for 1 hour. Digestion was stopped by adding Pefabic SC toa final of 4 mM. The digested homogenate was spiked in IP bufferfollowed by standard IP (lane 3, 7,11,15). (4) Nuclease digestion:scrapie hamster brain homogenate was treated with Benzonase® nuclease at100 U/mL at 37 C for 1 hour. Digestion was stopped by adding EDTA to afinal of 10 mM. The digested homogenate was spiked in IP buffer followedby standard IP (lane 4, 8,12,16).

[0082]FIG. 4. Immunocapture of PrP^(Sc) from brains of sCJD and vCJD byOCD4. The OCD4 conjugated beads were used to immunoprecipitate PrP inclarified brain homogenates from patients affected by either sCJD orvCJD, and two unaffected subjects (normal controls). Theimmunoprecipitates were then analyzed by SDS-PAGE (12% gel) and Westernblotting using the anti-PrP antibody 3F4. OCD4 specifically capturesPrP^(Sc) in brains of sCJD (lane 1) and vCJD (lane 3) but not PrP^(C) innormal brains (lanes 5 and 7). OCD4 captured PrP from sCJD and vCJDbrains are authentic PrP^(Sc) since treatment with PK (50 ug/ml for 1 hat 37° C.) generates the PK-resistant core PrP^(res) fragments (lanes 2and 4). Right panel. The clarified brain homogenates from sCJD, vCJD andnormal controls were incubated in the absence (−) or presence (+) of PK(50 ug/ml for 1 h at 37° C.). After the PK digestion was terminated,samples were directly loaded onto SDS-PAGE gels (12%) and analyzed onWestern blots using the 3F4 antibody. Brain tissues of sCJD type 2 andvCJD are well-characterized TSE reference materials from WHO.

[0083]FIG. 5. The OCD4 based capture immunoassay for PrP^(Sc) in brainsof vCJD patients. Brain homogenates (10 ul) from 10 cases of vCJD(v1-v10) were used in the OCD4/3F4 capture immunoprecipitation assay.The experiment was conducted at the NCJDSU, UK, using kindly providedvCJD cases (case numbers: 95/052(1), 96/045(2), 97/049(3), 98/063(4),98/148(5), 98/154(6), 99/082(7), 99/090(8), 00/066(9), 00/101(10)).

[0084]FIG. 6. Immunocapture of both the full-length PrP^(Sc) and thePK-resistant core fragments by OCD4. Aliquots of brain homogenates, fromthree vCJD cases were used in this experiment and conducted at NCJDSU,UK. The first aliquot was digested with PK (+PK). The second aliquot wasdigested with PK and then was subjected to immunoprecipitation by theOCD4 (+PK+OCD4). The third aliquot underwent direct immunoprecipitationby OCD4 without the PK treatment (−PK+OCD4). All above samples wereanalyzed on Western blots using the 3F4 antibody. The PK-res PrPfragments (lanes 1-5) were recovered in the OCD4 immunoprecipitates(lanes 6-10). The full-length PrP^(Sc) in untreated samples could beefficiently captured by OCD4 as well (lanes 11-15). Note that the inputvolume of the 10% brain homogenates was varied in some cases for betterresolution of the PrP bands.

[0085]FIG. 7. OCD4 Immunocapture of PK-resistant PrP in cVJD spleen.Spleen lysate was prepared from a case of vCJD (case number 96/045 and98/148, provided by NCJDSU at Edinburgh) using 10% homogenate followedby brief centrifuge to remove debris. PK treatment was performed at 50ug/ml for 1 h at 37° C. before the reaction was terminated by 10 mMPefabloc. Aliquots of each 100 ul of digested spleen lysate were eitherpelleted by centrifugation at 14,000 Xg for 1 h at 4° C. (lane 1), orwere subjected to immunoprecipitation with OCD4 (lane 2 and 4) and withan unrelated mAb (lane 3 and 5, as control for non-specific binding).Detection of PK-resistant PrP in spleen was done on Western blots usingthe 3F4 antibody.

[0086]FIG. 8. OCD4 immunocapture of BSE PrP^(sc). Brain lysates (10%,w/v) from either BSE or bovine control (CON) were prepared byhomogenization in lysis buffer followed by brief centrifugation.Aliquots of clarified brain lysates (1 ul each) were used inimmunoprecipitation (IP) before (lanes 1 and 2) or after (lanes 3 and 4)PK digestion (50 ug/ml for 1 h at 37° C.). Additional aliquots (1 ul)without IP and PK digestion (lanes 5 and 6) served as reference fortotal input. All samples were then separated by SDS-PAGE and weredetected on Western blots using the 6H4 antibody capable of recognizingbovine PrP. OCD4 immuoprecipitated PrP only from BSE brain in theabsence or presence of PK. This experiment was conducted at the P3facility of the Veterinary Laboratories Agency in Waybridge, Londonwhere brain tissues of BSE-affected and normal cattle were kindlyprovided.

[0087]FIG. 9. OCD4 Immunocapture of PrP^(Sc) in scrapie sheep.Immunoprecipitation by OCD4 of PrP from brains of natural scrapie (Sc)and normal sheep control (N) before and after treatment with PK (50ug/ml for 1 h at 37° C.). Experiments were performed as described in themain text. Immunoprecipitates on the OCD4 conjugated beads were probedon Western blots with the 6H4 antibody on Western blots. Each assay used1 ul of 10% brain homogenates.

[0088]FIG. 10. Effective immunocapture of spiked PrP^(Sc) in humanplasma by OCD4. The spike material (S) used was 1 uL of 5% homogenate ofscrapie hamster brain. Standard PrP^(sc) immunocapture in IP buffer (1mL) was shown in lanes 1 and 8. For lanes 2-4, the spike (1 uL) wasadded to 0.6 ml of three normal human plasma preparations A, B, and C(0.6 ml plasma/S) with the addition of 400 uL IP buffer. Lanes 5-7represent the non-spiked plasma aliquots (0.6 ml plasma) with 0.4 mL IPbuffer. For lanes 9-11, 5 mL each of plasma A, B, and C werepreincubated with the OCD4 beads followed by brief washes of the beadsin PBS. The plasma treated beads were then incubated with the spike (1uL) in 1 mL of IP buffer (5 mL plasma→S). Standard immunoprecipitationby the OCD conjugated beads was done in a total volume of 1 mL followedby Western blotting using the 3F4 antibody as described in the maintext. As compared to the input control (lanes 1 and 8), significantrecapture of spiked PrP^(Sc) by OCD4 was achieved in plasma present inlarge excess (600 uL plasma vs. 1 uL spike) (lanes 2-4). Moreover, thefact that preincubation of OCD4 conjugated beads with large volume ofnormal plasma did not compromise or block its binding ability to capturePrP^(Sc) as indicated in lane 9-11, exclude the possibility of potentialOCD4 inhibitors present in human plasma. OCD4 did not capture PrP^(C)from human plasma (lanes 5-7).

DETAILED DESCRIPTION OF THE INVENTION

[0089] The term “sample” as used herein, refers to any substance, whichmay contain the analyte of interest. A sample can be biological fluid,such as whole blood or whole blood components including red blood cells,white blood cells, platelets, serum and plasma, ascites, urine,cerebrospinal fluid, and other constituents of the body which maycontain the analyte of interest, such as brain homogenate. Optionally,samples may be obtained from water, soil, and vegetation. The term“patient” as used herein, refers to humans and/or animals.

[0090] Various immunoassay protocols are known and could be applied tothe present invention. The assay can be carried out using any enzymelabel which can be attached to the anti-prion antibody to form alabelled ligand. Enzymes such as oxidases, e.g., glucose oxidase,peroxidases, e.g., horseradish peroxidase (HRP), alkaline phosphataseand galactosidases are preferred labels. It is within the skill of oneof ordinary skill in the art to determine a suitable substrate for agiven label. The substrate can be a material which is directly actedupon by the enzyme label or a material that is involved in a series ofreactions which involve enzymatic reaction of the label. Other labelsand means for detection could be for example, a ligand, nucleotide, orbiotin. Detection of the labeled antibody could be by various methodsincluding enzyme amplification with polymeric conjugates and immuno PCR.

[0091] The following examples are given to illustrate but not limit thescope of the invention.

[0092] Brain Homogenate Preparation:

[0093] Normal and scrapie hamster brain lysate were obtained fromBaltimore Research and Education Foundation as 10% whole brain tissuehomogenate in PBS (w/v). The lysate was further treated by adding 1/10volume of 10X detergent homogenate buffer, composed of 5% sodiumdeoxycolate and 5% Igpal CA-630 (equivalent to NP-40) in PBS, incubatingat 4 C for 1 hr., followed by centrifugation at 1000 g for 10 minutes.The resulting supernatant was collected and used in the assay.

[0094] Normal and BSE bovine brain tissue were provided by VeterinaryLaboratories Agency (VLA), UK. Normal and scrapie sheep brain tissuewere provided by Animal Disease Research Unit of USDA, USA. Normal humanbrain tissue were provided by National Prion Disease PathologySurveillance Center (NPDPSC), USA. Human sCJD and vCJD brain tissue wereprovided by NPDPSC and National CJD Surveillance Unit (NCJDSU), UK.Brain tissue was processed the same way (or similar) as hamster brainhomogenate preparation.

[0095] Anti-DNA Antibodies and DNA Binding Protein:

[0096] Monoclonal antibodies obtained from commercial sources were (1)murine monoclonal antibody recognizing ss-, ds-DNA, subclass IgM, Cat#12403 and subclass IgG2b, Cat# 12404 from QED Bioscience, (2) murinemonoclonal antibody recognizing ds-DNA, clone AE-2, subclass IgG3, Cat#2660-2308 and murine monoclonal antibody recognizing ss-, ds-, clone49/4A1, subclass Ig2b, Cat# 2660-2316 from Biogenesis. The immunogensused to raise these antibodies were Calf thymus DNA and nuclei from RajiBurkitts lymphoma Cells as indicated by manufactures. Additionalmonoclonal antibodies from other than commercial source were alsoevaluated. Single Stranded Binding Protein (SSB) from E.coli purchasedfrom Sigma (Sigma, Mo., USA, Cat.# S3917).

[0097] Preparation of Immunogens:

[0098] The immunogen used to generate anti-DNA antibodies was nuclearDNA extracted from mammalian cells based upon known protocols (Sambrook1989) and monoclonal antibodies were also generated standard protocol(Yokoyama 2001). The antibodies were screened by ELISA using the coatedDNA immunogen.

[0099] Various cell lines are available to use in the identifiedprotocols. For example, OCD4 and antibody AE-2 were generated from fromDNA extracted from Raji Burkitts lymphoma cell line. One skilled in theart would certainly recognize however that other known cell lines andmethods are available. For example, 49/4A1, 12403 and 12404 weregenerated from DNA extracted from calf thymus DNA and then screened byELISA using the coated DNA immunogen.

[0100] OCD4 (100 μg of purified IgG) was conjugated to 7×10⁸ tosylactivated superparamagnetic beads (Dynabeads M-280, Dynal Co.) in 1 mlof phosphate-buffered saline (PBS) at 37° C. for 20 h (29). The OCD4conjugated beads were incubated with 0.1% bovine serum albumin (BSA) in(PBS) to block non-specific binding. The prepared OCD4 beads were stablefor at least 3 months at 4° C. Brain homogenate (10%, w/v) was preparedin lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodiumdeoxycholate, 10 mM Tris-HCI, pH 7.5, and a cocktail of proteaseinhibitors), followed by centrifugation at 3,000×g for 10 min at 4° C.to remove debris. Immunoprecipitation was performed using 5 μl of theclarified homogenate and 10 μl of OCD4 conjugated beads in 1 ml of IPbuffer containing 0.1% Tween-20 and 0.1% Nonidet P-40 in PBS, pH 7.5.After incubation with constant mixing for 2 h at room temperature, OCD4beads were attracted to the sidewall of the plastic tubes by externalmagnetic force, allowing easy removal of all unbound materials in thesolution. After three washes in the same buffer, OCD4 beads werecollected and were boiled for 10 min in SDS sample buffer (3% sodiumdodecyl sulfate (SDS), 2 mM EDTA, 10% glycerol, 50 mM Tris-HCI, pH 6.8).The eluted proteins were separated by 15% SDS-PAGE (15% Tris-glycinepre-cast gel, Bio-Rad), and were than analyzed by Western blottingeither with anti-PrP antibody 3F4 recognizing residues 109-112 (17) or6H4 recognizing residues 145-152 (14). PrP bands were visualized onKodak X-ray film using enhanced chemiluminescence.

[0101] Conjugation of Antibody and Protein to Magnetic Beads:

[0102] 0.35 mL Dynabeads® M-280 Tosylactivated (Dynal Biotech, N.Y.,USA, Cat.# 142.03/04) were washed twice with PBS and the beads isolatedfrom buffer with the magnet (Dynal Magnetic Particle Concentrator, MPC).100 ug of purified antibody or protein in 1 mL PBS was added to thewashed beads. Incubation with rotation was performed at 37 C for 18-20hours. The beads were isolated from the buffer with the MPC, washedtwice with 1 ml PBS (0.1% BSA), and rotated for 5 minutes at roomtemperature while washing. The antibody-conjugated beads were thenblocked for 3-4 hours, 37° C. with 0.2 M Tris-HCI, pH 8.0, containing0.1% BSA. The beads were subsequently washed 2 times with 1 ml PBS (0.1%BSA) and once with 1 ml PBS (0.1% BSA, 1% Tween 20) incubating each timefor 10 minutes at room temp. The beads were then washed once with 1 mlPBS (0.1%BSA) and then stored in 1 ml PBS (0.05% sodium azide) at 4° C.

[0103] Proteinase K Digestion and Benzonase® Nuclease Digestion:

[0104] Conditions for the PK digestion of brain lysate: Brain homogenatewas suspended in PBS buffer with or without non-ionic detergent. Thetotal homogenate protein concentration was no more than 2.5 mg/mL. PK(Roche Diagnostics, IN, USA, Cat.# 1373196) was added to a finalconcentration of 50 ug/mL. Incubation was at 37 C for 0.5 to 1 hour.Digestion was stopped by adding Pefabloc SC (Roche Diagnostics, IN, USA,Cat.# 1585916) to a final concentration of 4 mM. Conditions for theBenzonase® Nuclease digestion of brain lysate: Brain homogenate wassuspended in Tris-HCI buffer, with or without non-ionic detergent,containing 2 mM Mg⁺⁺. Total homogenate protein concentration was no morethan 2.5 mg/mL. Nuclease (CN Biosciences, CA, USA, Cat.# 70664) wasadded to a final concentration of 100 U/mL. Incubation was at 37 C for0.5 to 1 hour. Digestion was stopped by adding EDTA to a finalconcentration of 10 mM.

[0105] Immunoprecipitation (IP). Non-reducing Electrophoresis andImmunoblot Detection of PrP^(sc):

[0106] Anti-DNA antibody conjugated magnetic beads were used to capturePrP^(Sc) from brain homogenate by immunoprecipitation. The IP procedureconsists of the following protocol: mix 100 uL antibody conjugated beadswith 1-5 uL of brain homogenate in a total of 1 mL IP buffer (3% Tween20and 3% Igpal CA-630 in PBS) and incubate at 25 C for 2.5 hours withrotation→Separate beads using MPC device and wash beads 3 times of 30second vortexing with IP wash buffer (2% Tween20 and 2% Igpal CA-630 inPBS)→Elute captured PrP^(Sc) by heating beads with NuPAGE sample bufferfor 10-15 minutes. The eluted sample from IP capture were loaded onto a4-12% NuPAGE® Bis-Tris Gel (Invitrogen, CA, USA, Cat.# NP0302) andsubjected to non-reducing electrophoresis at 200V for 45 minutes. Theimmunoblot procedure was perfomed as follows: transfer separatedproteins in the gel to a 0.2 um PVDF membrane (Invitrogen, Cat# LC2002)at 30V for 60 minutes→4 Block the membrane with Blocker™ Casein in TBS(0.05% Tween20) (Pierce Chemical Corp., IL, USA, Cat.# 37532) either at25 C for 1 hour with shaking or at 4 C overnight. →As primary antibody,use 3F4 (Signet, MA, USA, Cat.# 9620-02) at 1:3000 dilution to detecthamster and human PrP^(Sc) or use 6H4 (Prionics AG, Switzerland, Cat.#01-011) at 1:5000 dilution or to detect bovine and sheep PrP^(Sc)respectively. Incubate the membrane with diluted primary antibody in 10%Blocker™ Casein in TBST buffer (25 mM Tri-Cl, 0.2M NaCl, 0.2% Tween20,pH 8.0) at 25 C for 1 hour with shaking. →Wash 3×5 minutes with TBSTbuffer with shaking. →Incubate membrane with horseradish peroxidaseconjugated goat polyclonal anti-mouse IgG (H+L) (Jackson ImmunoResearchLaboratories, PA, USA, Cat.# 115-035-003) at 1:10,000 to 1:30,000dilution in 50% Blocker™ Casein in TBST buffer at 25 C for 1 hour withshaking. →Wash 6×5 minutes with TBST buffer with shaking. →Add ECLchemiluminescence substrate (Amersham Biosciences, NJ, USA, Cat.#RPN2109) or SuperSignal West Dura chemiluminescence substrate (Pierce)on membrane to develop for 5 minutes. →Take image by exposure either toBio Max MR-2 film (Kodak, NY, USA) or to the ChemiDoc Gel DocumentationSystem (Bio-Rad, CA, USA).

[0107] Vaccine and Therapetic Uses

[0108] Another aspect of the invention is directed toward therapeuticuses of the anti-nucleic acid antibodies as a therapeutic use. Animalmodels can be infected, for example with vCJD. One skilled in the artwould then inject the animal with anti-nucliec acid antibodies in orderto bind and neutralize the infectious prions. The result would be areduction or elimination of the disease.

[0109] Advantages.

[0110] The present invention uses anti-DNA to capture PrP^(Sc) byrecognition of high affinity associated nucleic acid in the nucleicacid::PrP^(Sc) complex. Because the tight association of nucleic acidonly to PrP^(Sc) and not to PrP^(C), the present invention provided anon-intrusive means for the detection of PrP^(Sc) while no PK digestionor other protein modification procedure required. It is anticipated thatthe mild conditions will preserve the original structure andconformation of the pathogenic prion protein, thereby offeringopportunity to determine the presence of true PrP^(Sc) while minimizingthe generation of PrP^(Sc) due to harsh sample treatment.

[0111] Provided evidence that Benzonase nuclease digestion does notcompromise selective anti-DNA binding to nucleic acid::PrP^(Sc),including limited endonuclease treatment in sample preparation orcomprised in sample buffer could eliminate the interference ofendogenous nucleic acid interference.

[0112] The use of anti-DNA antibodies offer advantages in that theydisplay the binding specificity but can also be easily handled in directcoating to a solid phase as well as be conjugated to link to signalgiven reagents such as horseradish peroxidase (HRP), or to be adoptedinto other desired diagnosis assay format.

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We claim:
 1. A method for discriminating between infectious andnoninfectious prions comprising: first contacting a sample with ananti-nucleic acid antibody, then adding a prion specific antibody toform a complex between the anti-nucleic acid antibody, prion and prionspecific antibody, and detecting the complexes.
 2. A method fordiagnosing transmissible spongiform encephalopathies in a patientcomprising: drawing a sample from a patient, contacting a sample with ananti-nucleic acid antibody, then adding a prion specific antibody toform a complex between the anti-nucleic acid antibody, prion and prionspecific antibody, and detecting the complexes, whereby detecting thecomplexes provides an indication of transmissible spongiformencephalopathies in a patient.
 3. An immunoassay for detectinginfectious prions comprising: providing a solid support having boundthereto an anti-nucleic acid antibody, contacting the solid support witha sample, washing the support to remove any unbound sample, contactingthe solid support with a prion specific antibody, and carrying out adetection step to determine if prions are bound to the solid support. 4.A kit for the detection of infectious prions comprising a solid supporthaving bound thereto an anti-nucleic acid antibody, and a labeled prionspecific antibody.
 5. An immunoassay for detecting infectious prionscomprising: providing a solid support coated with an agent to bind ananti-nucleic acid antibody contacting the solid support with a sample,washing the support to remove any unbound sample, contacting the solidsupport with a prion specific antibody, and carrying out a detectionstep to determine if prions are bound to the solid support.
 6. Theimmunoassay claimed in claim 5 wherein the agent is avidin orstreptavidin and the anti-nucleic acid antibody has been biotinylated.7. A vaccine composition comprising anti-nucleic acid antibodies and apharmaceutically acceptable carrier.
 8. A method of treating a priondisease in a patient comprising administering a therapeuticallyeffective amount of a vaccine composition comprising anti-nucleic acidantibodies and a pharmaceutically acceptable carrier.
 9. A method ofinducing neutralized infectious prions in a patient susceptible to orsuffering from a prion disease comprising administering atherapeutically effective amount of a vaccine composition comprisinganti-nucleic acid antibodies and a pharmaceutically acceptable carrier.